This invention is generally in the field of production of polyhydroxyalkanoates (PHAs) by genetic engineering of bacteria.
Synthesis of PHA polymers containing the monomer 4-hydroxybutyrate (4HB), such as poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (PHB4HB) (Doi, 1995, Macromol Symp. 98:585-99) or 4-hydroxyvalerate and 4-hydroxyhexanoate containing PHA polyesters have been described, for example, in Valentin et al., 1992, Appl. Microbial. Biotechnol, 36:507-14 and Valentin et al., 1994, Appl. Microbiol. Biotechnol. 40:710-16. Production of PHB4HB, for example, has been accomplished by feeding glucose and 4HB or a substrate that is converted to 4-hydroxybutyrate to Ralstonia eutropha (Kunioka, et al., 1988, Polym. Commun. 29:174; Doi, et al., 1990, Int. J. Biol. Macromol. 12:106; Nakamura, et al., 1992, Macromolecules 25:423), to Alcaligenes latus (Hiramitsu, et al., 1993, Biotechnol. Lett. 15461), to Pseudomnonas acidovorans (Kimura, et al., 1992, Biotechnol. Lett. 14:445), and to Comamonas acidovorans (Saito & Doi, 1994, Int. J. Biol. Macromol. 16:18). Substrates that are converted to 4HB include 1,4-butanediol, 1,6-hexanediol, 1,8-octanediol, 1,10-decanediol, 1,12-dodecanediol and gamma-butyrolactone. The PHB4HB copolymers can be produced with a range of monomer compositions which provide a range of polymer properties. In particular, as the amount of 4HB increases above 15 wt. %, the melting temperature (Tm) decreases below 130° C. and the elongation to break increases above 400% (Saito, et al., 1996, Polym. Int. 39:169).
It would be highly advantageous, however, to develop more cost effective ways of producing PHAs containing 4HB by biological systems. For economic production of PHA, several factors are critical, including substrate costs, fermentation time, and efficiency of downstream processing. A general characteristic of the wild type PHA-producing bacteria is that their growth rate is low, they are often difficult to break open and their amenity to genetic engineering is limited. Therefore, it would be desirable to develop transgenic organisms that provide improved economics of PHA production.
The production of the copolymer PHB4HB in recombinant E. coli has been described (e.g., PCT WO 00/011188 by Huisman et al.; PCT WO 98/39453 by Hein et al.). A range of novel biologically produced 4HB polymers produced in recombinant E. coli have been described by Skraly and Peoples (e.g., PCT WO 99/61624). In these studies only the Huisman reference demonstrated the incorporation of small amounts of 4HB co-monomer from 1,4-butanediol. It would be highly advantageous to develop genetically engineered systems capable of the production of a range of 4HB copolymers and poly-4HB homopolymer using 1,4-butanediol as the source of the 4HB monomer.
It is therefore an object of the present invention to provide improved recombinant systems and methods for the production of PHAs, such as PHAs containing the 4HB monomer, using a variety of simple sugars and alcohols as substrates.